Cancers contain altered methylation patterns that result in aberrant expression of critical genes. Hypermethylation turns off expression of genes required to regulate normal growth while hypomethylation allows for inappropriate expression of genes that allow cells to proliferate. Aberrant promoter hypermethylation occurs at the 5-position of cytosine within the CpG dinucleotide. Gardiner-Garden, M., et al., J. Mol. Biol., 196(2): 261-82 (1987). It inactivates the expression of critical genes that are involved in tumor suppression, DNA repair, control of tumor metastasis, and invasion. Feinberg, A. P., et al., Nature, 301: 89-92 (1983); Jones, P. A., et al., Nat. Rev. Genet., 3(6): 415-28 (2002). There is a great need in both basic and clinical research to identify promoter DNA methylation status with high efficiency and accuracy for diseases diagnoses and prognoses.
Various methods have been developed for the study of promoter DNA methylation status of known genes. Laird P. W., Nature Review Cancer, 3: 253-266 (2003). These methods can generally be grouped into two categories: methylation-sensitive restriction endonuclease assays and sodium bisulfite conversion based approaches.
Methylation-Sensitive Restriction Endonuclease Digestion Methods
The enzymatic digestion method traditionally relies on the inability of methylation-sensitive enzymes to cleave restriction sites containing methylated CpG dinucleotides. Genomic DNAs are incubated with the proper restriction endonucleases and the presence and absence of the cleaved DNA fragments can then be identified by Southern hybridization. This method is not only capable of analyzing the methylation status of individual known genomic region, but also allows the global examination of CpG island methylation status. However, the disadvantage is that large quantity of high molecular weight genomic DNA is required to begin with Issa, J. P., et al., Nature Genetic, 7(4): 536-40 (1994). This method is suitable for the study where a high percentage of alleles of interest are methylated, such as imprinted genes and X chromosome inactivated genes; this method is not suitable for clinical applications where the quantity and quality of the genomic DNA resource can be a limiting factor.
To circumvent the requirement of large quantity of high molecular weight genomic DNA, a more sensitive approach based on the combination of methylation-sensitive restriction endonuclease digestion and the polymerase chain reaction has also been introduced. Singer-Sam, J., et al., Nucleic Acids Res., 18(3): 687 (1990), Singer-Sam, J., et al., Mol. Cell. Biol., 10(9): 4987-9 (1990). Oligonucleotide polymerase chain reaction (“PCR”) primers have been designed flanking the restriction endonuclease site, and PCR amplification is performed after the enzymatic digestion. A methylated restriction endonuclease site results in the presence of the proper PCR product. On the other hand, PCR template can be cleaved by the endonuclease if the restriction site is unmethylated. The credibility of this method depends on the complete digestion of unmethylated DNA by the restriction endonuclease. The problem is exacerbated by the fact that the sample DNA is often limited, and it is difficult to drive endonuclease digestions to completion. Thus, it is sometimes difficult to determine whether PCR amplicons result from incomplete digestion (i.e. false positives) or from those of low abundance methylation sites (i.e. true positives). Restriction enzyme techniques are based on removing the unmethylated DNA, and assuming that PCR amplification of the remaining DNA arises because it was methylated, and consequently the method is susceptible to false positives arising from incomplete removal of unmethylated DNA.
Sodium Bisulfite Based Chemical Conversion Approaches
Chemical conversion of cytosines to uracils using bisulfite can be used to study DNA methylation. 5-methylcytosines are resistant to conversion and deamination only occurs on unmethylated cytosines. Frommer, M., et al., Proc. Natl. Acad. Sci. USA, 89(5): 1827-31 (1992). Bisulfite can be quantitatively added to the 5-6 double bonds of cytosine if there is no methyl group on the 5 position. Bisulfite addition renders the cytosine susceptible to hydrolytic deamination; subsequent elimination of the bisulfite results in the formation of uracil. Voss, K. O., et al., Anal. Chem., 70(18): 3818-3823 (1998). One strand of the modified DNA sequences can then be PCR amplified and sequenced. However, due to stromal cell contamination in a typical clinical sample, direct sequencing without cloning the PCR products reduces the sensitivity of the technique. It requires about 25% of the alleles to be methylated for accurate detection. Myohanen, S., et al., DNA Sequence, 5: 1-8 (1994).
The development of methylation-specific PCR (MSP) has allowed the sensitive and specific study of low abundance methylation sequences. Herman, J. G., et al., Proc. Natl. Acad. Sci. USA, 93(18): 9821-6 (1996). MSP relies upon chemical modification of DNA using bisulfite, the specific designed PCR primers that are complementary to the bisulfite modified DNA template. The MSP specific primers are designed across a CpG rich area within a promoter sequence. Typically, more than three CpG sites have to be included in the oligonucleotide sequences. Two sets of MSP PCR primers are designed, one set of the MSP primers has the sequence to perfectly hybridize to the complementary strand of the bisulfite-treated methylated DNA sequence with methyl-cytosines residing on the CpG sites. The other set of the MSP primers is only designed to perfectly hybridize to the complementary strand of the bisulfite-treated DNA sequence in the absence of methylated cytosine. Consequently, the MSP specific PCR products only results from the DNA template which contains methyl-cytosines.
There are three major difficulties with this approach. The design of MSP primers requires sufficient numbers of methylated cytosines to be present in the primer sequence to ensure the selection capability. It may not be sufficiently sensitive to distinguish partial methylated sequences from fully methylated one. In addition, this assay analyzes one gene at a time, and both sets of MSP primers have different annealing temperatures which may further slowdown its throughput. Finally, bisulfite treatment of DNA often nicks the DNA (i.e. destroys the backbone chain) as it is also converting unmethylated cytosines to uracil. Conditions which assure that all unmethylated cytosines are converted to uracil may also destroy the DNA. Conditions which assure that sufficient DNA remains intact may not assure that all unmethylated cytosines are converted to uracil. Thus, absence of a band may be the consequence of destroying too much of the starting DNA and, consequently, insufficient amplification, leading to a false negative result. Likewise, presence of a band may be the consequence of incomplete conversion of unmethylated cytosine to uracil, allowing for primer binding at an unmethylated site, and leading to a false positive result. Therefore, there is an urgent need to develop a high-throughput assay that can detect methylation status in virtually any gene sequence.
The present invention is directed to meeting this need.